An update on the pharmacokinetics and pharmacodynamics of alisertib, a selective Aurora kinase A inhibitor.

Human Aurora kinases, including Aurora kinase A (AURKA), B (AURKB), and C (AURKC), play an essential role in mitotic events such as monitoring of the mitotic checkpoint, creation of bipolar mitotic spindle and alignment of centrosomes on it, also regulating centrosome separation, bio-orientation of chromosomes and cytokinesis. AURKA and AURKB are key regulators of mitosis and centrosome via polymerizing microfilaments and controlling chromatid segregation. In particular, AURKA plays critical roles in the regulation of mitotic entry, centrosome function, bipolar spindle assembly, and chromosome segregation. AURKA has been found to be overexpressed in various solid and haematological cancers and has been linked with poor prognosis. Its important role in cancer initiation, growth, and metastasis has brought the focus to search for potent and selective AURKA inhibitors for cancer treatment. MLN8237, also known as alisertib, is one selective AURKA inhibitor that has shown remarkable anticancer effects in preclinical studies. Alisertib exhibits favourable pharmacokinetic properties. Alisertib has generally showed good partial response rates of 4-52% and good safety profiles in Phase I and II trials when it is solely administered as well as combined with cytotoxic chemotherapeutic drugs. Recently, the multicentre, randomized Phase III study of alisertib in patients with relapsed or refractory peripheral T-cell lymphoma has been discontinued due to unsatisfactory efficacy. The low risk of side effects, accessibility, and effectiveness of alisertib makes it a new promising anticancer therapy and further mechanistic and clinical studies are warranted.

Three surgical planes identified in laparoscopic complete mesocolic excision for right-sided colon cancer.

BACKGROUND: Complete mesocolic excision provides a correct anatomical plane for colon cancer surgery. However, manifestation of the surgical plane during laparoscopic complete mesocolic excision versus in computed tomography images remains to be examined. METHODS: Patients who underwent laparoscopic complete mesocolic excision for right-sided colon cancer underwent an abdominal computed tomography scan. The spatial relationship of the intraoperative surgical planes were examined, and then computed tomography reconstruction methods were applied. The resulting images were analyzed. RESULTS: In 44 right-sided colon cancer patients, the surgical plane for laparoscopic complete mesocolic excision was found to be composed of three surgical planes that were identified by computed tomography imaging with cross-sectional multiplanar reconstruction, maximum intensity projection, and volume reconstruction. For the operations performed, the mean bleeding volume was 73±32.3 ml and the mean number of harvested lymph nodes was 22±9.7. The follow-up period ranged from 6-40 months (mean 21.2), and only two patients had distant metastases. CONCLUSIONS: The laparoscopic complete mesocolic excision surgical plane for right-sided colon cancer is composed of three surgical planes. When these surgical planes were identified, laparoscopic complete mesocolic excision was a safe and effective procedure for the resection of colon cancer.

Impact of physiological, pathological and environmental factors on the expression and activity of human cytochrome P450 2D6 and implications in precision medicine.

With only 1.3-4.3% in total hepatic CYP content, human CYP2D6 can metabolize more than 160 drugs. It is a highly polymorphic enzyme and subject to marked inhibition by a number of drugs, causing a large interindividual variability in drug clearance and drug response and drug-drug interactions. The expression and activity of CYP2D6 are regulated by a number of physiological, pathological and environmental factors at transcriptional, post-transcriptional, translational and epigenetic levels. DNA hypermethylation and histone modifications can repress the expression of CYP2D6. Hepatocyte nuclear factor-4α binds to a directly repeated element in the promoter of CYP2D6 and thus regulates the expression of CYP2D6. Small heterodimer partner represses hepatocyte nuclear factor-4α-mediated transactivation of CYP2D6. GW4064, a farnesoid X receptor agonist, decreases hepatic CYP2D6 expression and activity while increasing small heterodimer partner expression and its recruitment to the CYP2D6 promoter. The genotypes are key determinants of interindividual variability in CYP2D6 expression and activity. Recent genome-wide association studies have identified a large number of genes that can regulate CYP2D6. Pregnancy induces CYP2D6 via unknown mechanisms. Renal or liver diseases, smoking and alcohol use have minor to moderate effects only on CYP2D6 activity. Unlike CYP1 and 3 and other CYP2 members, CYP2D6 is resistant to typical inducers such as rifampin, phenobarbital and dexamethasone. Post-translational modifications such as phosphorylation of CYP2D6 Ser135 have been observed, but the functional impact is unknown. Further functional and validation studies are needed to clarify the role of nuclear receptors, epigenetic factors and other factors in the regulation of CYP2D6.

Targeting Na⁺/K⁺ -translocating adenosine triphosphatase in cancer treatment.

The Na(+) /K(+) -translocating adenosine triphosphatase (ATPase) transports sodium and potassium across the plasma membrane and represents a potential target in cancer chemotherapy. Na(+) /K(+) -ATPase belongs to the P-type ATPase family (also known as E1-E2 ATPase), which is involved in transporting certain ions, metals, and lipids across the plasma membrane of mammalian cells. In humans, the Na(+) /K(+) -ATPase is a binary complex of an α-subunit that has four isoforms (α1 -α4 ) and a ß-subunit that has three isoforms (ß1 -ß3 ). This review aims to update our knowledge on the role of Na(+) /K(+) -ATPase in cancer development and metastasis, as well as on how Na(+) /K(+) -ATPase inhibitors kill tumour cells. The Na(+) /K(+) -ATPase has been found to be associated with cancer initiation, growth, development, and metastasis. Cardiac glycosides have exhibited anticancer effects in cell-based and mouse studies via inhibition of the Na(+) /K(+) -ATPase and other mechanisms. Na(+) /K(+) -ATPase inhibitors may kill cancer cells via induction of apoptosis and autophagy, radical oxygen species production, and cell cycle arrest. They also modulate multiple signalling pathways that regulate cancer cell survival and death, which contributes to their antiproliferative activities in cancer cells. The clinical evidence supporting the use of Na(+) /K(+) -ATPase inhibitors as anticancer drugs is weak. Several phase I and phase II clinical trials with digoxin, Anvirzel, and huachansu (an intravenous formulated extract of the venom of the wild toad), either alone or more often in combination with other anticancer agents, have shown acceptable safety profiles but limited efficacy in cancer patients. Well-designed randomized clinical trials with reasonable sample sizes are certainly warranted to confirm the efficacy and safety of cardiac glycosides for the treatment of cancer.

Clinical pharmacology of dipeptidyl peptidase 4 inhibitors indicated for the treatment of type 2 diabetes mellitus.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a class of oral antidiabetic drugs that improve glycaemic control without causing weight gain or increasing hypoglycaemic risk in patients with type 2 diabetes mellitus (T2DM). The eight available DPP-4 inhibitors, including alogliptin, anagliptin, gemigliptin, linagliptin, saxagliptin, sitagliptin, teneligliptin, and vildagliptin, are small molecules used orally with identical mechanism of action and similar safety profiles in patients with T2DM. DPP-4 inhibitors may be used as monotherapy or in double or triple combination with other oral glucose-lowering agents such as metformin, thiazolidinediones, or sulfonylureas. Although DPP-4 inhibitors have the same mode of action, they differ by some important pharmacokinetic and pharmacodynamic properties that may be clinically relevant in some patients. The main differences between the eight gliptins include: potency, target selectivity, oral bioavailability, elimination half-life, binding to plasma proteins, metabolic pathways, formation of active metabolite(s), main excretion routes, dosage adjustment for renal and liver insufficiency, and potential drug-drug interactions. The off-target inhibition of selective DPP-4 inhibitors is responsible for multiorgan toxicities such as immune dysfunction, impaired healing, and skin reactions. As a drug class, the DPP-4 inhibitors have become accepted in clinical practice due to their excellent tolerability profile, with a low risk of hypoglycaemia, a neutral effect on body weight, and once-daily dosing. It is unknown if DPP-4 inhibitors can prevent disease progression. More clinical studies are needed to validate the optimal regimens of DPP-4 inhibitors for the management of T2DM when their potential toxicities are closely monitored.

An update on the clinical pharmacology of the dipeptidyl peptidase 4 inhibitor alogliptin used for the treatment of type 2 diabetes mellitus.

Alogliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor that is a class of relatively new oral hypoglycaemic drugs used in patients with type 2 diabetes (T2DM), can be used as monotherapy or in combination with other anti-diabetic agents, including metformin, pioglitazone, sulfonylureas and insulin with a considerable therapeutic effect. Alogliptin exhibits favorable pharmacokinetic and pharmacodynamic profiles in humans. Alogliptin is mainly metabolized by cytochrome P450 (CYP2D6) and CYP3A4. Dose reduction is recommended for patients with moderate or worse renal impairment. Side effects of alogliptin include nasopharyngitis, upper-respiratory tract infections and headache. Hypoglycaemia is seen in about 1.5% of the T2DM patients. Rare but severe adverse reactions such as acute pancreatitis, serious hypersensitivity including anaphylaxis, angioedema and severe cutaneous reactions such as Stevens-Johnson syndrome have been reported from post-marketing monitoring. Pharmacokinetic interactions have not been observed between alogliptin and other drugs including glyburide, metformin, pioglitazone, insulin and warfarin. The present review aimed to update the clinical information on pharmacodynamics, pharmacokinetics, adverse effects and drug interactions, and to discuss the future directions of alogliptin.

Alisertib Induces Cell Cycle Arrest, Apoptosis, Autophagy and Suppresses EMT in HT29 and Caco-2 Cells.

Colorectal cancer (CRC) is one of the most common malignancies worldwide with substantial mortality and morbidity. Alisertib (ALS) is a selective Aurora kinase A (AURKA) inhibitor with unclear effect and molecular interactome on CRC. This study aimed to evaluate the molecular interactome and anticancer effect of ALS and explore the underlying mechanisms in HT29 and Caco-2 cells. ALS markedly arrested cells in G2/M phase in both cell lines, accompanied by remarkable alterations in the expression level of key cell cycle regulators. ALS induced apoptosis in HT29 and Caco-2 cells through mitochondrial and death receptor pathways. ALS also induced autophagy in HT29 and Caco-2 cells, with the suppression of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), but activation of 5' AMP-activated protein kinase (AMPK) signaling pathways. There was a differential modulating effect of ALS on p38 MAPK signaling pathway in both cell lines. Moreover, induction or inhibition of autophagy modulated basal and ALS-induced apoptosis in both cell lines. ALS potently suppressed epithelial to mesenchymal transition (EMT) in HT29 and Caco-2 cells. Collectively, it suggests that induction of cell cycle arrest, promotion of apoptosis and autophagy, and suppression of EMT involving mitochondrial, death receptor, PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways contribute to the cancer cell killing effect of ALS on CRC cells.

Computational and in vitro studies on the inhibitory effects of herbal compounds on human cytochrome P450 1A2.

Human CYP1A2 is an important enzyme for drug metabolism and procarcinogen activation. This study aimed to explore the binding mode of ligands with CYP1A2 and to screen potential inhibitors from a library of herbal compounds using computational and in vitro approaches. The heme prosthetic group and six residues (Thr124, Phe125, Phe226, Phe260, Gly316, and Ala317) in the active site of CYP1A2 were identified as important residues for ligand binding using the LIGPLOT program. Ala317 in helix I immediately above heme was highly conserved in most human CYPs with known crystal structures. In molecular docking, 19 of the 56 herbal compounds examined were identified as potential inhibitors of CYP1A2. Up to 21 of the 56 herbal compounds were hit by the pharmacophore model of CYP1A2 inhibitors developed and validated in this study. In the in vitro inhibition study, 8 herbal compounds were identified as moderate to potent inhibitors of CYP1A2. Five of the 8 herbal compounds predicted to be potential inhibitors were confirmed as CYP1A2 inhibitors in the in vitro study. A combination of computational and in vitro approaches, represent a useful tool to identify potential inhibitors for CYP1A2 from herbal compounds.

Therapeutic effect of transplanting magnetically labeled bone marrow stromal stem cells in a liver injury rat model with 70%-hepatectomy.

BACKGROUND: There are only few reports about the use of bone marrow stromal stem cells (BMSCs) for the treatment of traumatic liver injury. This study aimed to study the therapeutic effect of fluorescence-labeled BMSCs administered to rats subject to traumatic liver injury. MATERIAL/METHODS: Male SD rats with a 70% resection of the liver were injected with feridex-labeled BMSCs which could be induced to functional hepatocytes in vitro. Liver function was assayed and the liver scanned by 1.5-T MRI at 12 hrs and on days 1, 3, 5, 7, and 14 post-operation. The pathological changes of liver sections were monitored. RESULTS: The serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, direct bilirubin, and total bilirubin in the transplantation group were significantly lower than the control group. The MRI showed rats of the transplantation group had an oval low signal area at 12 hr after operation; the low signal range gradually expanded and the signal intensity gradually decreased over 14 days after operation. The low signal range in the control group disappeared 12 hr after the operation. After Prussian blue staining, rats of the transplantation group contained blue granules with no significant hypertrophy or edema in hepatocytes, while the control group showed no blue granules with significant hypertrophy and edema. CONCLUSIONS: The BMSCs transplanted into the injured rat liver gradually migrate to the surrounding liver tissue and partially repair the liver surgical injury in rats. BMSCs may represent an effective therapeutic approach for acute liver injury.

Osthole, a herbal compound, alleviates nucleus pulposus-evoked nociceptive responses through the suppression of overexpression of acid-sensing ion channel 3 (ASIC3) in rat dorsal root ganglion.

BACKGROUND: Osthole (Ost), a natural coumarin derivative, has been shown to inhibit many pro-inflammatory mediators and block voltage-gated Na+ channels. During inflammation, acidosis is an important pain inducer which activates nociceptors by gating depolarizing cationic channels, such as acid-sensing ion channel 3 (ASIC3). The aim of this study was to examine the effects of Ost on nucleus pulposus-evoked nociceptive responses and ASIC3 over-expression in the rat dorsal root ganglion, and to investigate the possible mechanism. MATERIAL/METHODS: Radicular pain was generated with application of nucleus pulposus (NP) to nerve root. Mechanical allodynia was evaluated using von Frey filaments with logarithmically incremental rigidity to calculate the 50% probability thresholds for mechanical paw withdrawal. ASIC3 protein expression in dorsal root ganglions (DRGs) was assessed with Western blot and immunohistochemistry. Membrane potential (MP) shift of DRG neurons induced by ASIC3-sensitive acid (pH6.5) was determined by DiBAC(4) (3) fluorescence intensity (F.I.). RESULTS: The NP-evoked mechanical hyperalgesia model showed allodynia for 3 weeks, and ASIC3 expression was up-regulated in DRG neurons, reaching peak on Day 7. Epidural administration of Ost induced a remarkable and prolonged antinociceptive effect, accompanied by an inhibition of over-expressed ASIC3 protein and of abnormal shift of MP. Amiloride (Ami), an antagonist of ASIC3, strengthened the antinociceptive effect of Ost. CONCLUSIONS: Up-regulation of ASIC3 expression may be associated with NP-evoked mechanical hyperalgesia. A single epidural injection of Ost decreased ASIC3 expression in DGR neurons and the pain in the NP-evoked mechanical hyperalgesia model. Osthole may be of great benefit for preventing chronic pain status often seen in lumbar disc herniation (LDH).

Interaction of herbal compounds with biological targets: a case study with berberine.

Berberine is one of the main alkaloids found in the Chinese herb Huang lian (Rhizoma Coptidis), which has been reported to have multiple pharmacological activities. This study aimed to analyze the molecular targets of berberine based on literature data followed by a pathway analysis using the PANTHER program. PANTHER analysis of berberine targets showed that the most classes of molecular functions include receptor binding, kinase activity, protein binding, transcription activity, DNA binding, and kinase regulator activity. Based on the biological process classification of in vitro berberine targets, those targets related to signal transduction, intracellular signalling cascade, cell surface receptor-linked signal transduction, cell motion, cell cycle control, immunity system process, and protein metabolic process are most frequently involved. In addition, berberine was found to interact with a mixture of biological pathways, such as Alzheimer's disease-presenilin and -secretase pathways, angiogenesis, apoptosis signalling pathway, FAS signalling pathway, Hungtington disease, inflammation mediated by chemokine and cytokine signalling pathways, interleukin signalling pathway, and p53 pathways. We also explored the possible mechanism of action for the anti-diabetic effect of berberine. Further studies are warranted to elucidate the mechanisms of action of berberine using systems biology approach.

[Adenovirus vector mediated human 14-3-3 γ gene transfer protects dopaminergic cells against rotenone-induced injury].

OBJECTIVE: To explore the protective effect of human 14-3-3 γ gene transfer on dopaminergic cells against rotenone-induced injury. METHODS: Adenovirus vector carrying the gene of 14-3-3 γ (Ad/14-3-3 γ) was employed to transfect PC12 cells. Then the cells were exposed to rotenone as a model of Parkinson's disease. Methyl thiazolyl tetrazolium (MTT) was used to assay the viability of PC12 cells. The 4',6-diamidino-2-phenylindole (DAPI) staining was used to analyze the apoptotic ratio of PC12 cells among the groups of control, Ad/14-3-3 γ, Ad-null and Rotenone. And high performance liquid chromatography (HPLC) was performed to detect the secreting functions of PC12 cells. The aggregates of α-Synuclein protein were detected under confocal microscopy. RESULTS: MTT showed that the cell absorbance A(570) of Ad/14-3-3 γ group (0.46 ± 0.09) was higher than that of Ad-null group (0.19 ± 0.08) and rotenone group (0.16 ± 0.07), but lower than that of normal control (0.63 ± 0.11), (all P < 0.01); HPLC-ECD showed that the levels of dopamine (189 ± 11) ng/ml and noradrenalin (55 ± 8) ng/ml in the culture fluid of Ad/14-3-3 γ group were higher than those of Ad-null group (79 ± 12, 38 ± 7) ng/ml and rotenone group (81 ± 13, 39 ± 7) ng/ml (all P < 0.01). DAPI staining showed that cell apoptosis ratio of group Ad/14-3-3 γ (27% ± 64%) was lower than that of group Ad-null (53% ± 10%) and rotenone group (56% ± 12%, P < 0.01), but higher than that of control group (10% ± 5%, P < 0.01). Under confocal microscopy, the aggregates of α-synuclein protein in PC12 cells were detected more in Ad-null group and rotenone group than that in Ad/14-3-3 γ group. CONCLUSION: Gene transfer of Ad/14-3-3 γ has a protective effect on dopaminergic cells against rotenone-induced injury.

Combination of Body Mass Index and Fasting Blood Glucose Improved Predictive Value of New-Onset Prediabetes or Diabetes After Acute Pancreatitis: A Retrospective Cohort Study.

OBJECTIVES: We sought to evaluate whether combining body mass index (BMI) and fasting blood glucose (FBG) can refine the predictive value of new-onset prediabetes/diabetes after acute pancreatitis (NODAP). METHODS: In this retrospective cohort study, we used Kaplan-Meier analysis to compare differences in the NODAP rate among 492 patients with different BMI or FBG levels, or with the combination of these 2 factors mentioned above. RESULTS: In all, 153 of 492 (31.1%) eligible patients finally developed NODAP. According to univariate and multivariate analyses, BMI (hazard ratio, 2.075; 95% confidence interval, 1.408-3.060; P < 0.001) and FBG (hazard ratio, 2.544; 95% confidence interval, 1.748-3.710; P < 0.001) were important predictors of the incidence of NODAP. Subsequently, we divided 492 eligible patients into 3 groups according to the median BMI and FBG values, and found that the NODAP rate in the high-risk group was significantly higher than that in the medium-risk group ( P = 0.018) or the low-risk group ( P < 0.001). CONCLUSIONS: Body mass index and FBG are independent predictors of NODAP. The combination of BMI and FBG can refine the prediction of NODAP and identify candidates for clinical prevention.

Transcription profiles of skin and head kidney from goldfish suffering hemorrhagic septicemia with an emphasis on the TLR signaling pathway.

Hemorrhagic septicemia is an acute, highly fatal disease that affects goldfish (Carassius auratus). To gain a better understanding of related immune genes, the transcriptomes of the skin and head kidney of goldfish suffering hemorrhagic septicemia were sequenced, assembled, and characterized. Based on functional annotation, an extensive and diverse catalog of expressed genes were identified in both the skin and head kidney. As two different organs, pair-wise comparison identified 122/77 unigenes up/down-regulated (two-fold change with P<0.05) in the skin and head kidney. Most genes of the immune pathways were expressed and isolated in both skin and head kidney, including interferon (IFN) transcription factors 1-10 and Toll-like receptors (TLRs). Interferon regulatory factor 3 (IRF3), a key IFN transcription factor, was up-regulated at the transcriptional level by polyriboinosinic: polyribocytidylic acid (poly I:C) challenge and regulated the IFN response by increasing the activity of IFN-ß and IFN-stimulated response element (ISRE)-containing promoter. This study will benefit the identification and understanding of novel genes that play important roles in the immunological reactions of fish suffering from hemorrhagic septicemia.

Favorable effects of VEGF gene transfer on a rat model of Parkinson disease using adeno-associated viral vectors.

Vascular endothelial growth factor (VEGF) is a specific angiogenic peptide, which has been identified to play a critical role in neurodegeneration, and has beneficial effects on neurons. In this study, we investigated whether neurodegeneration in a rat model of Parkinson disease could be prevented by VEGF gene transfer mediated by adeno-associated virus (AAV) vectors. Our results demonstrated that a single injection of a VEGF-expressing AAV vector into striatum improved the rotational behavior of rat Parkinson disease models, and promoted the survival of dopaminergic neurons and fibers. Meanwhile, AAV-VEGF injection significantly increased the reactive astrocytes and the levels of glial cell line-derived neurotrophic factor in striatum, but did not induce extra angiogenesis and remarkable disorder of blood-brain barrier. We thus conclude that intrastriatal delivery of VEGF gene mediated by AAV has favorable effects on the dopaminergic neurons in a rat Parkinson disease model.

[The tracking of allogenic grafted rat bone marrow stem cells in rat liver and their role on repairing injured liver].

OBJECTIVE: To study the location, immigration of allogenic grafted Feridex-labeled rat bone marrow stem cells (BMSCs) in chemically-induced acute injured livers and their role on repairing the injured liver function. METHODS: The rat models of chemically-induced acute hepatic injury established with CCl4 Feridex-labeled BMSCs were injected into the injured livers. Magnetic resonance imaging (MRI) examination was conducted on rat livers, the levels of ALT, AST and Fe3+ in the serum and hepatic tissues were studied 6 h before and 6 h, 1 w and 5 w after injection. RESULTS: Cellular necrosis, congestion in the hepatic sinusoid and infiltration of inflammatory cells were seen in the model livers. Above 90 percent of the cells were Feridex-labeled BMSCs positive by prussian blue staining and iron particles were found in their endochylema through electron microscopy. MRI examination at the sequence of SE-T2WI showed remarkably low signal changes 6 h after injecting Feridex-labeled BMSCs and the site with signal changes gradually expanded 1 and 5 w after injection. Comparatively, the changes of low signal images at each time point in the injured livers were more obvious than those of the controls at all time points, respectively. Simultaneously, pathological injuries in the livers were ameliorated and the levels of ALT and AST in serums declined: these changes in the Feridex-labeled BMSCs group were more obvious than those in the non-Feridex-labeled BMSCs group. Uniformly, there were no significant differences between the Feridex-labeled BMSCs group and the non-Feridex-labeled BMSCs group in view of histopathological examination and serological examination (including ALT, AST, Fe3+ levels) at all time points. CONCLUSIONS: The liver function in the model of chemically-induced acutely-injured liver may be repaired by BMSCs implantation. Traced by MRI, BMSCs in the injured liver of rats disperse at a higher rate than in the normally fed ones.

Inhibition of Aurora A Kinase by Alisertib Induces Autophagy and Cell Cycle Arrest and Increases Chemosensitivity in Human Hepatocellular Carcinoma HepG2 Cells.

BACKGROUND: Aurora A kinase represent a feasible target in cancer therapy. OBJECTIVE: To evaluate the proteomic response of human liver carcinoma cells to alisertib (ALS) and identify the molecular targets of ALS, we examined the effects of ALS on the proliferation, cell cycle, autophagy, apoptosis, and chemosensitivity in HepG2 cells. METHOD: The stable-isotope labeling by amino acids in cell culture (SILAC) based quantitative proteomic study was performed to evaluate the proteomic response to ALS. Cell cycle distribution and apoptosis were assessed using flow cytometry and autophagy was determined using flow cytometry and confocal microscopy. RESULTS: Our SILAC proteomic study showed that ALS regulated the expression of 914 proteins, with 407 molecules being up-regulated and 507 molecules being down-regulated in HepG2 cells. Ingenuity pathway analysis (IPA) and KEGG pathway analysis identified 146 and 32 signaling pathways were regulated by ALS, respectively, which were associated with cell survival, programmed cell death, and nutrition-energy metabolism. Subsequently, the verification experiments showed that ALS remarkably arrested HepG2 cells in G2/M phase and led to an accumulation of aneuploidy via regulating the expression of key cell cycle regulators. ALS induced a marked autophagy in a concentration- and time-dependent manner via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Autophagy inhibition promoted the pro-apoptotic effect of ALS, indicating a cyto-protective role of ALS-induced autophagy. ALS increased the chemosensitivity of HepG2 cells to cisplatin and doxorubicin. CONCLUSION: Taken together, ALS induces autophagy and cell cycle arrest in HepG2 cells via PI3K/Akt/mTOR-mediated pathway. Autophagy inhibition may promote the anticancer effect of ALS and sensitize the chemotherapy in HepG2 cells.

Curcumin enhances the effects of irinotecan on colorectal cancer cells through the generation of reactive oxygen species and activation of the endoplasmic reticulum stress pathway.

Although initially effective against metastatic colorectal cancer (CRC), irinotecan-based chemotherapy leads to resistance and adverse toxicity. Curcumin is well known for its anti-cancer effects in many cancers, including CRC. Here, we describe reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress as important mechanisms by which curcumin enhances irinotecan's effects on CRC cells. CRC cell lines were treated with curcumin and/or irinotecan for 24 h, and then evaluated using cell proliferation assays, cell apoptosis assays, cell cycle analysis, intracellular Ca2+ measurements, ROS measurements and immunoblotting for key ER stress-related proteins. We found that cell viability was inhibited and apoptosis was increased, accompanied by ROS generation and ER stress activation in CRC cells treated with curcumin alone or in combination with irinotecan. Blocking ROS production attenuated the expression of two markers of ER stress: binding of immunoglobulin protein (BIP) and CCAAT/enhancer-binding protein homologous protein (CHOP). Blocking CHOP expression using RNA interference also inhibited ROS generation. These results demonstrated that curcumin could enhance the effects of irinotecan on CRC cells by inhibiting cell viability and inducing cell cycle arrest and apoptosis, and that these effects may be mediated, in part, by ROS generation and activation of the ER stress pathway.

[Adenovirus mediated vascular endothelial growth factor gene transfer protects dopaminergic neurons in Parkinson's disease: experiment with rats].

OBJECTIVE: To evaluate the protective effect of adenovirus mediated vascular endothelial growth factor 165 (VEGF165) gene transfer on dopaminergic neurons in Parkinson disease (PD). METHODS: Adenovirus vector coding VEGF165 (Ad-VEGF165) was injected into the striate bodies of 16 SD rats, and adenovirus Ad-LacZ was injected into 25 rats and phosphate-buffered saline (PBS) was injected into 16 rats as controls. Then 6-hydroxydopamine (6-OHDA) was injected to establish PD model. X-gal staining was used to detect the expression of the report gene LacZ in the brain of the Ad-LacZ group 3 d, 2 w, and 6 w after injection, 3 rats in each time point. RT-PCR was used to detect the VEGF165 mRNA expression in the striate body of the rats of the 3 groups 2 weeks after injection, 3 rats for each group. Western blotting was performed to check the protein expression of VEGF165 in the striate bodies of the rats of the 3 groups 2 weeks after injection, 3 rats for each group. A certain numbers of rats in each group underwent rotational behavior analysis 1, 2, and 6 weeks after the 6-OHDA injection. Immunohistochemistry was used to examine the number of tyrosine hydroxylase (TH) positive neuron, density of TH-positive fiber in striate body and black substance, laminin-positive vessel density, and glial fibrillary acidic protein (GFAP) positive glial cells. High performance liquid chromatography-electric-chemical discharge (HPLC-ECD) was performed to detect the contents of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum. RESULTS: Beta-gal was expressed in the striate bodies of all Ad-LacZ transfected rats, showing the successful transfection of LacZ gene. The mRNA expression and protein expression of VEGF165 in the striate body were significantly higher in the Ad-VEGF165 group than in the other 2 groups. The apomorphine-induced rotation number in the Ad-VEGF165 group was 8.3 turns/min +/- 8.7 turns/min 1 week after the transfection, then gradually decreased, and became 5.0 turns/min +/- 4.4 turns/min 6 weeks after. The rotation numbers of the Ad-LacZ and PBS group were 14.7 turns/min +/- 11.2 turns/min and 13.9 turns/min +/- 8.3 turns/min respectively 1 week after the injection, then increased gradually, and became 20.2 turns/min +/- 13.6 turns/min and 21.8 turns/min +/- 11.8 turns/min respectively 6 weeks later, all significantly higher than those of the Ad-LacZ group (all P < 0.01). The ratios of TH-positive cells in the black substance in the transfected side was 0.42 +/- 0.11, and the density of fibers in the striate body of the transfected side to that of the contralateral side was 0.56 +/- 0.10 in the Ad-VEGF165 group, both significantly higher than those of the Ad-LacZ group (0.20 +/- 0.10 and 0.28 +/- 0.09) and PBS group (0.22 +/- 0.13 and 0.24 +/- 0.08), (all P < 0.01). The ratio of laminin-positive vessel density of the transfected side to that of the contralateral side in the Ad-VEGF165 group was 2.09 +/- 0.42, and the ratio of GFAP-positive glial cells of the striate body of the transfected side to that of the contralateral side was 2.77 +/- 1.21 in the Ad-VEGF165 group, both significantly higher than those in the Ad-LacZ group (1.01 +/- 0.16 and 1.64 +/- 0.28) and the PBS group (1.04 +/- 0.09 and 1.56 +/- 0.62) (P < 0.01 and 0.05). HPLC-ECD showed that the contents of DA, HAV, and DOPAC of the striate body at the destroyed side in the Ad-VEGF165 group were all significantly higher than those in the other 2 groups (all P < 0.01). The ratios to the DA, DOPAC, and HVA of the destroyed side striate body to those of the contralateral side in the Ad-VEGF165 group was 0.35 +/- 0.11, 0.46 +/- 0.09, and 0.38 +/- 0.09 respectively, all significantly higher than those in the Ad-LacZ group (0.17 +/- 0.15, 0.21 +/- 0.07, and 0.16 +/- 0.05) and PBS group (0.19 +/- 0.06, 0.20 +/- 0.09, 0.14 +/- 0.03) (all P < 0.01). CONCLUSION: Gene transfer of Ad-VEGF165 has a protective effect on the dopaminergic neurons of PD. The proliferation of vessels and glial cells induced by VEGF may involve in the process of neuroprotection to the dopaminergic neurons of PD.

A Comparison of Non-Human Primate Cytochrome P450 2D Members and the Implication in Drug Discovery.

BACKGROUND: Non-human primates are valuable animal models in drug discovery and biomedical research. Human CYP2D6 accounts for 1.3-4.3% of total hepatic CYP content in the liver, but is involved in the metabolism of more than 150 drugs. With the advancement of genomic sequencing and annotation, a panel of CYP2D genes have been cloned from non-human primates. This review highlights the similarities and differences of these CYP2D genes non-human primates. METHODS: We conducted a structured PubMed search using a focused review question and proper inclusion/exclusion criteria. The quality of retrieved papers was assessed and briefed using standard tools and expert knowledge. RESULTS: Most studies on CYP expression in non-human primates have been carried out in the cynomolgus and Rhesus monkeys. Deduced amino acid sequences of primate CYP2D cDNAs share high sequence identity (93-96%) with human CYP2D6. The chimpanzee genome has CYP2D6 and 2D7 but bonobos only contain CYP2D6. The CYP2D6 gene is located on chromosome 22 in the chimpanzee genome (human CYP2D6 maps to chromosome 22q13.1), and on chromosome 10 in the genome of the Rhesus monkey. Cynomolgus monkey CYP2D17 and Japanese monkey 2D29 metabolize bufuralol and dextromethorphan. CYP2D17 metabolizes bufuralol and dextromethorphan, whereas CYP2D29 metabolizes bufuralol and debrisoquine. In addition, quinidine inhibits both cynomolgus monkey CYP2D17 and Japanese monkey 2D29. CONCLUSION: The CYP2D members from non-human primates show differential genomic contexts, catalytic activities toward substrates and inhibitory profiles. Further studies are warranted to elucidate the structural and functional features of CYP2D members in non-human primates and thus offer a solid base for the application of these animals in drug discovery.